RNA editing of ion channels and receptors has important consequences to their function. RNA editing is a post-transcriptional modification of pre-mRNA performed by an enzyme that converts adenosine into inosine (AI), which is interpreted as guanosine by the translational machinery. Thereby, RNA editing provides a substantial expansion of the genetic pool of an organism. As might be intuitively expected, AI conversions often target critical positions of the encoded protein, where changes in function are essential for the physiology of a cell. Over the past six years, we have become increasingly interested in understanding how nature functionally tunes membrane proteins by RNA editing. [unreadable] RNA editing allows multiple protein products from a single gene. This increase in genomic capability does not appear to be random, rather it targets regions of a protein that are functionally important. The classical example is in the GluRB subunit of glutamate-gated ion channels, important for fast excitatory synaptic transmission in the central nervous system. Editing underlies the conversion of glutamine to arginine in the channelfs pore (Sommer et al., 1991), which renders the receptor impermeable to calcium ions (Kohler et al., 1993). Although editing seems to be common among membrane proteins (Hoopengardner et al., 2003), the functional consequences of editing had been explored in only a few examples (Kohler et al., 1993; Burns et al., 1997; Patton et al., 1997; Wang et al., 2000; Berg et al., 2001; Rosenthal and Bezanilla, 2002; Bhalla et al., 2004). We have joined efforts with the laboratory of Josh Rosenthal to approach the subject of RNA editing of membrane proteins in a comprehensive manner. We have already characterized the functional consequences of an I to V conversion within the intracellular cavity of the human KV1.1 channel (Bhalla et al., 2004). In this case, RNA editing targets fast inactivation, an additional gating mechanism of KV1.1 channels. This study also provided the foundation to ask previously inaccessible questions about the interactions between the inactivation particle and the ion conduction pore. Because fast inactivation occurs by the direct occlusion of the permeation pathway by an inactivating particle (Hoshi et al., 1990; Zagotta et al., 1990; Demo and Yellen, 1991; Zhou et al., 2001a), we are asking which amino acids from this particle interact with the core of the channel, and what is the specific mechanisms by which fast inactivation is altered by the I to V conversion . Our preliminary observations suggest that the end of the N-terminus can actually enter deep into the intracellular cavity and interact in close proximity with the edited position. [unreadable] Transporters are essential for ion channel function because they provide and maintain the ionic gradient that allows ions to diffuse through channels. Recently, by comparing genomic and cDNAs sequences, new targets of RNA editing have been identified, among them, proteins involved in ion homeostasis (Stapleton et al., 2006). Interestingly, there is no report in the literature of how RNA editing might alter the function of any transporter. We are taking advantage of the apparent high levels of editing in squid (Patton et al., 1997; Rosenthal and Bezanilla, 2002) to examine RNA editing in transporters, initially focusing our attention on the Na+/K+ pump, a transporter that I have studied over the past 15 years, and the Na+/Ca2+ exchanger. At present, we have cloned the full-length cDNA and genomic DNA for both transporters from squid, and we have found at least four potential RNA editing sites in the Na+/K+ pump and six in the Na+/Ca2+ exchanger. We have been successful in expressing the genomic and edited constructs of these transporters in Xenopus oocytes, which will allow us to characterize the functional consequences of the editing events. We have already uncovered potentially interesting functional consequences in both transporters. In the Na+/K+ pump, RNA editing seems to produce an increase in the apparent affinity for intracellular ATP. In the exchanger, regulation by intracellular Ca2+ appears to be altered by editing. Our goal is to develop a mechanistic understanding of how editing influence the function of these transporters, hopefully to the level of details we are acquiring in KV1.1 channels. [unreadable] As we proceed with these projects, we have exploited the uniqueness of our system to ask biologically significant questions. For example, once we cloned and expressed the SqNaK ATPase, the first pump from a marine osmoconformer to be characterized, we were able to ask and explain how the squid pump can operate efficiently when exposed to almost half a molar of extracellular Na+. The mammalian pump would be almost completely inhibited at negative resting potentials by the high Na+ of sea water because the pumpfs turnover rate is inhibited as external Na+ ions are driven back to their binding sites (by voltage) through physically narrow access channels (Gadsby et al., 1993). We discovered that the electrostatics of the external mouth of the squid pump are such that the local Na+ at the entrance to the Na+ permeation pathway is the same as for the mammalian pump.